The effects of lipid in direct contact with a GPCR are of profound importance for signaling. Three essential conditions need to be met in order to successfully carry out the rigorous characterization of GPCR signaling properties, namely (i) the lipid environment surrounding the GPCR has to be controlled with high precision; (ii) ligand and the intracellular signaling partners must have simultaneous access to the extracellular and intracellular receptor faces, respectively; and (iii) the oligomeric state of the receptor has to be well defined. The above criteria are satisfied by the nanodisc technology developed by Sligar and colleagues. We have investigated the effect of phospholipid head group charges on agonist and G protein interaction with NTS1 as a first step towards understanding the role of annular lipids in signaling. All experiments were conducted using the zwitterionic lipid POPC, a mixture of POPC/POPG, or the negatively charged lipid POPG for the reconstitution of NTS1 into nanodiscs. We found that the nanodisc lipid compositions did not substantially modulate agonist binding to NTS1. In contrast to NT binding, Gq protein activation was dramatically affected by the nature of the lipid present in the respective nanodiscs. The apparent affinity of Gq and nucleotide exchange rates increased with the presence of POPG in NTS1-nanodiscs. Compared to NTS1-POPC-nanodiscs, normalized nucleotide exchange rates were increased by 1 and 2 orders of magnitude for NTS1-POPC/POPG- and NTS1-POPG-nanodiscs, respectively. We have suggested that the strong effect of the negatively charged POPG on G protein affinity and nucleotide exchange rates may arise from a combination of the following mechanisms: Partitioning of G protein components to the receptor-nanodiscs, specific interactions between lipid and receptor residues, and local net charges at the NTS1/lipid interface. The latter aspect is intriguing, as a negative surface potential leads to accumulation of hydronium ions at the NTS1/lipid interface, possibly favoring the protonation of Glu166 of the conserved E(D)RY motif in NTS1 and hence the productive interaction with the G protein. Future work will address the interaction of G protein-coupled receptor kinases and arrestins with NTS1. Taken together, these experiments will provide insight into the preference of NTS1 with its interacting signaling molecules.